Lipids are first synthesized as saturated fatty acids and double bonds are introduced post-synthetically by oxygen-dependent enzymes known as fatty acid desaturases, in a process that is initiated by abstraction of hydrogen from a methylene group. Fatty acid desaturases are divided into soluble and integral membrane classes, which may have been evolved independently (Shanklin J, Somerville C., “Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs”, Proc Natl Acad Sci USA 1991; 88:2510-4). The acyl-ACP desaturases are soluble enzymes found in the plastids of higher plants, whereas the more widespread class of integral membrane acyl-CoA desaturases is found in endomembrane systems in prokaryotes and eukaryotes (Shanklin J, Cahoon E B., “Desaturation and Related Modifications of Fatty Acids1”, Annu Rev Plant Physiol Plant Mol Biol 1998; 49:611-41). Fatty acid desaturases in each class are closely related homologs based on their amino acid sequences, and yet perform highly regio- and stereo-selective reactions on long-chain fatty acids composed of essentially equivalent methylene chains that lack distinguishing landmarks close to the site of desaturation. As pointed out by Nobel Laureate Dr. Konrad Bloch, this region- and stereo-specific removal of hydrogen “would seem to approach the limits of the discriminatory power of enzymes” (Bloch K., “Enzymatic synthesis of monounsaturated fatty acids”, Accounts of Chemical Research 1969; 2:193-202).
The membrane class of desaturases consists of enzymes with c5, c6, c9, c12 or ω3 regio-selectivity. Mammalian cells possess c5, c6 and c9, but lack c12 and ω3 desaturases (Berquin I M, Edwards I J, Kridel S J, Chen Y Q, “Polyunsaturated fatty acid metabolism in prostate cancer”, Cancer Metastasis Rev 2011; 30:295-309, and Chen Y Q, Edwards I J, Kridel S J, Thornburg T, Berquin I M., “Dietary fat-gene interactions in cancer”, Cancer Metastasis Rev 2007; 26:535-51). Mortierella alpina belongs to the subphylum of Mucoromycotina (Hibbett D S, Binder M, Bischoff J F, et al., “A higher-level phylogenetic classification of the Fungi”, Mycol Res 2007; 111:509-47). It can produce lipids up to 50% of its dry weight. We have recently characterized M. alpina genome (Wang L, Chen W, Feng Y, et al., “Genome Characterization of the Oleaginous Fungus Mortierella alpine”, PLoS One 2011; 6:e28319) which encodes one c5, two c6, three c9, one c12 and one ω3 desaturase. Therefore, M. alpina has all known regio-selective groups of membrane desaturases.
We have expressed M. alpina c9, c12 and ω3 desaturases (FADS9-I, FADS12 and FADS15) in the methylotrophic yeast Pichia pastoris, purified the recombinant proteins and determined their enzymatic activities.